New results from this laboratory indicate four novel relationships between the ms2i6A-37 tRNA modification and mutagenesis in E. coli. These findings have lead to the hypothesis that undermodification of ms2i6A-37 to i6A-37 or A-37 acts as a "physiological switch" to increase spontaneous mutation frequency. Such undermodification occurs during iron limitation of miaA+ bacteria or in miaA (formerly trpX) mutants defective in ms2i6A-37 formation. Thus, this specific tRNA undermodification allows cells to increase their mutation frequencies in response to certain environmental stresses without exposure to mutagens. The long-term objective of this five-year proposal is to evaluate critically the latter hypothesis and to characterize the genetic components, targets, and physiology of this tRNA- linked switch. To meet this goal, the following six Specific aims will be completed. (I) Transposon insertion mutations will be isolated in genes involved in ms2i6A-37 biosynthesis, other than miaA. According to the above hypothesis, these new mutants should know increased spontaneous mutation frequencies. (II) The steps in DNA repair or the fidelity of replication will be identified that are directly or indirectly hindered by ms2i6A-37 tRNA undermodification. A variety of physiological and genetic characterizations will be performed to meet this aim. (III) Analysis will be continued of the structure and regulation of miaA, which likely forms a complex operon with mutL. Emphasis will be placed on promising genetic approaches to isolate regulatory mutants and on determining why 2- aminopurine induces chromosomal miaA transcription. (IV) Mutation frequencies will be measured in bacteria stressed by conditions that cause ms2i6A-37 tRNA undermodification, other than iron limitation. According to the above hypothesis, physiological stress conditions, such as cysteine deprivation, should increase spontaneous mutation frequency. (V) Biochemical and genetic analyses will be performed on the mechanism that leads to ms2i6A-37 tRNA undermodification to determine whether active enzymatic removal of the ms2i6A-37 modification takes place. (VI) The structure and regulation of new genes identified in Aim I will be determined, since it is likely that these genes are members of complex operons and that they share modes of regulation with the complex mutL-miaA operon. Taken together, results from this proposal will provide important, basic information about the relationship between adaptation, mutation frequency and evolution, the genetic integration of cellular metabolism, the composition and control of complex operons, and the biosynthesis and functions of modified bases in tRNA molecules.